Human beta-alanine-pyruvate aminotransferase

ABSTRACT

The invention provides a human beta-alanine-pyruvate aminotransferase (HAPA) and polynucleotides which identify and encode HAPA. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HAPA.

This application is a divisional application of U.S. application Ser.No. 09/015,296, filed Jan. 29, 1998 now U.S. Pat. No. 6,103,471, all ofwhich application and patents are hereby incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman beta-alanine-pyruvate aminotransferase and to the use of thesesequences in the diagnosis, treatment, and prevention of diseasesassociated with immune disorders and cancer.

BACKGROUND OF THE INVENTION

Aminotransferases catalyze the transfer of an alpha-amino group from analpha-amino acid to an alpha-keto acid. These enzymes, also calledtransaminases, generally funnel alpha-amino groups from a variety ofamino acids to alpha-ketoglutarate for conversion into NH₄ ⁺. Aspartateaminotransferase, one of the most important of these enzymes, catalyzesthe transfer of the amino group of aspartate to alpha-ketoglutarate. Inmost vertebrates, NH₄ ⁺ is converted into urea, and is excreted.

In terrestrial vertebrates, urea is synthesized by the urea cycle. Oneof the nitrogen atoms of the urea synthesized by this pathway istransferred from the amino acid aspartate. The other nitrogen atom andthe carbon atom are derived from NH₄ ⁺ and CO₂. Ornithine is the carrierof these carbon and nitrogen atoms. Other reactions of the urea cyclelead to the synthesis of arginine from ornithine, an amino acid thatoccurs naturally as an intermediate in arginine biosynthesis. Alanineaminotransferase, which is also prevalent in mammalian tissue, catalyzesthe transfer of the amino group of alanine to alpha-ketoglutarate whichproducing pyruvate and glutamate. Glutamate is then oxadativelydeaminated, yielding NH₄ ⁺ and regenerating alpha-ketoglutarate. (See,e.g., Stryer, L., 1988 (3rd ed.). Freeman.) High levels of NH₄ ⁺ aretoxic to humans. The synthesis of urea in the liver is the major routeof removal of NH₄ ⁺, and a complete block of any of the steps of theurea cycle is usually fatal, because there is no known alternativepathway for the synthesis of urea. Inherited disorders caused by apartial block of each of the urea cycle reactions have been diagnosed.The most common condition is an elevated level of NH₄ ⁺ in the blood(hyperammonemia). A nearly total deficiency of any of the urea cycleenzymes results in coma or death shortly after birth.

The discovery of a new human beta-alanine-pyruvate aminotransferase andthe polynucleotides encoding it satisfies a need in the art by providingnew compositions which are useful in the diagnosis, treatment, andprevention of diseases associated with immune disorders and cancer.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, humanbeta-alanine-pyruvate aminotransferase (HAPA), comprising a sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant of HAPAhaving at least 90% amino acid identity to the sequence of SEQ ID NO:1or a fragment of SEQ ID NO:1. The invention also provides an isolatedand purified polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoincludes an isolated and purified polynucleotide variant having at least90% polynucleotide identity to the polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

Additionally, the invention provides a composition comprising apolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1. The invention further provides anisolated and purified polynucleotide which hybridizes under stringentconditions to the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, andan isolated and purified polynucleotide variant having at least 90%polynucleotide identity to the polynucleotide comprising the sequence ofSEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides anisolated and purified polynucleotide which is complementary to thepolynucleotide comprising the sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect,the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideencoding HAPA under conditions suitable for the expression of thepolypeptide; and (b) recovering the polypeptide from the host cellculture.

The invention also provides a pharmaceutical composition comprising asubstantially purified HAPA having the sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist to HAPA.

The invention also provides a method for treating or preventing animmune response, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist to HAPA.

The invention also provides a method for detecting a polynucleotideencoding HAPA in a biological sample containing nucleic acids, themethod comprising the steps of: (a) hybridizing the complement of thepolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleicacids of the biological sample, thereby forming a hybridization complex;and (b) detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding HAPA in the biological sample. In one aspect, the nucleic acidsof the biological sample are amplified by the polymerase chain reactionprior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of HAPA. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd., S. San Francisco, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among HAPA(3128715; SEQ ID NO:1) and beta-alanine-pyruvate aminotransferase fromrat (GI 1944136; SEQ ID NO:3), produced using the multisequencealignment program of LASERGENE software (DNASTAR Inc, Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

“HAPA,” as used herein, refers to the amino acid sequences ofsubstantially purified HAPA obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to HAPA, increases or prolongs the duration of the effect of HAPA.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of HAPA.

An “allele” or an “allelic sequence,” as these terms are used herein, isan alternative form of the gene encoding HAPA. Alleles may result fromat least one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

“Altered” nucleic acid sequences encoding HAPA, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same HAPA or apolypeptide with at least one functional characteristic of HAPA.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HAPA, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HAPA. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HAPA. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of HAPA isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments”, “immunogenic fragments”, or“antigenic fragments” refer to fragments of HAPA which are preferablyabout 5 to about 15 amino acids in length and which retain somebiological activity or immunological activity of HAPA. Where “amino acidsequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp. 1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which,when bound to HAPA, decreases the amount or the duration of the effectof the biological or immunological activity of HAPA. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HAPA.

As used herein, the term “antibody” refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHAPA polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term “antisense strand” is used in referenceto a nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

As used herein, the term “biologically active,” refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic HAPA, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding HAPA orfragments of HAPA may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase “consensus sequence,” as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR (PE Biosystems, Foster City, Calif.) in the 5′and/or the 3′ direction, and resequenced, or which has been assembledfrom the overlapping sequences of more than one Incyte Clone using acomputer program for fragment assembly, such as the GELVIEW FragmentAssembly system (GCG, Madison, Wis.). Some sequences have been bothextended and assembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HAPA, bynorthern analysis is indicative of the presence of nucleic acidsencoding HAPA in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding HAPA.

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of HAPA, of a polynucleotide sequence encoding HAPA, or ofa polynucleotide sequence complementary to a polynucleotide sequenceencoding HAPA. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (LASERGENE softwarepackage, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can createalignments between two or more sequences according to different methods,e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The Clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be calculated by theClustal Method, or by other methods known in the art, such as the JotunHein Method. (See, e.g., Hein, J. (1990) Methods in Enzymology183:626-645.) Identity between sequences can also be determined by othermethods known in the art, e.g., by varying hybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term “hybridization complex” as used herein, refersto a complex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀t or R₀t analysis) or formedbetween one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray,” as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term “modulate,” as it appears herein, refers to a change in theactivity of HAPA. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HAPA.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in reading frame, certain genetic elements, e.g.,repressor genes, are not contiguously linked to the encoded polypeptidebut still bind to operator sequences that control expression of thepolypeptide.

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimers,”“primers,” “oligomers,” and “probes,” as these terms are commonlydefined in the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HAPA,or fragments thereof, or HAPA itself may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “substantially purified,” as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

A “variant” of HAPA, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software.

THE INVENTION

The invention is based on the discovery of a new humanbeta-alanine-pyruvate aminotransferase (HAPA), the polynucleotidesencoding HAPA, and the use of these compositions for the diagnosis,treatment, or prevention of diseases associated with immune disordersand cancer.

Nucleic acids encoding the HAPA of the present invention were firstidentified in Incyte Clone 3128715 from the lung tumor cDNA library(LUNGTUT12) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones3128715 (LUNGTUT12), 3234316 (COLNUCT03), 2075940 (ISLTNOT01), 3186992(THYMNON04), 077724 (SYNORAB01), 1252424 (LUNGFET03), and 1620917(BRAITUT13).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,1D, and 1E. HAPA is 450 amino acids in length and has six potentialcasein kinase II phosphorylation sites at residues T₉₉, S₁₁₂, S₁₄₆,S₁₉₉, T₃₀₂, and T₄₃₄; three potential protein kinase C phosphorylationsites at residues S₂₂, T₁₇₃, and T₄₄₅; and one potentialaminotransferase class-III pyridoxal-phosphate attachment site betweenresidue F₂₄₃ and G₂₈₃, in which K₂₇₈ is the pyridoxal-phosphateattachment site. As shown in FIGS. 2A and 2B, HAPA has chemical andstructural homology with beta-alanine-pyruvate aminotransferase (GI1944136; SEQ ID NO:3). In particular, HAPA and beta-alanine-pyruvateaminotransferase from rat share 35% identity and the active lysineattachment site at residue K₂₇₈ in the pyridoxal-phosphate attachmentsite. Fragments of the nucleic acid sequence (SEQ ID NO:2) useful forhybridization experiments are from about C₉₇ to about G₁₀₈ and aboutA₂₂₀ to about G₂₃₅. Northern analysis shows the expression of thissequence in various libraries, at least 37% of which are immortalized orcancerous and at least 28% of which involve immune response.

The invention also encompasses HAPA variants. A preferred HAPA variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HAPA amino acid sequence, and which contains at least one functionalor structural characteristic of HAPA.

The invention also encompasses polynucleotides which encode HAPA. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an HAPA.

The invention also encompasses a variant of a polynucleotide sequenceencoding HAPA. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HAPA. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of HAPA.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HAPA, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HAPA, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HAPA and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HAPA under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HAPA or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HAPA and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodeHAPA and HAPA derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding HAPA or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; and Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.)

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taq DNApolymerase, thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE Amplification System (GIBCO/BRL, Gaithersburg,Md.). Preferably, the process is automated with machines such as theMICROLAB 2200 (Hamilton, Reno, Nev.), DNA ENGINE thermal cycler (PTC200;MJ Research, Watertown, Mass.) and the ABI PRISM sequencing systems (PEBiosystems).

The nucleic acid sequences encoding HAPA may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCRMethods Applic. 2:318-322.) In particular, genomic DNA is firstamplified in the presence of a primer complementary to a linker sequencewithin the vector and a primer specific to the region predicted toencode the gene. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia, T. etal. (1988) Nucleic Acids Res. 16:8186.) The primers may be designedusing commercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060.) Additionally, one mayuse PCR, nested primers, and PROMOTERFINDER libraries (Clontech, PaloAlto, Calif.) to walk genomic DNA. This process avoids the need toscreen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, PE Biosystetms), and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HAPA may be used in recombinant DNAmolecules to direct expression of HAPA, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHAPA.

As will be understood by those of skill in the art, it may beadvantageous to produce HAPA-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HAPA-encodingsequences for a variety of reasons including, but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HAPA may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HAPA activity, it may be useful toencode a chimeric HAPA protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HAPA encoding sequence and theheterologous protein sequence, so that HAPA may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HAPA may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp. Ser. (7):215-223,and Horn, T. et al. (1980) Nucl. Acids Symp. Ser. (7):225-232.)Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HAPA, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science269:202-204.) Automated synthesis may be achieved using the ABI 431APeptide synthesizer (PE Biosystems).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, e.g., Chiez,R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or by sequencing. (See, e.g., Creighton, T. (1983) ProteinsStructures and Molecular Properties, WH Freeman and Co., New York, N.Y.)Additionally, the amino acid sequence of HAPA, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active HAPA, the nucleotide sequencesencoding HAPA or derivatives thereof may be inserted into appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HAPA andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HAPA. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions, e.g., enhancers, promoters, and 5′ and 3′untranslated regions, of the vector and polynucleotide sequencesencoding HAPA which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters, e.g., hybrid lacZpromoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) orPSPORT1 plasmid (GIBCO/BRL), may be used. The baculovirus polyhedrinpromoter may be used in insect cells. Promoters or enhancers derivedfrom the genomes of plant cells (e.g., heat shock, RUBISCO, and storageprotein genes) or from plant viruses (e.g., viral promoters or leadersequences) may be cloned into the vector. In mammalian cell systems,promoters from mammalian genes or from mammalian viruses are preferable.If it is necessary to generate a cell line that contains multiple copiesof the sequence encoding HAPA, vectors based on SV40 or EBV may be usedwith an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HAPA. For example, when largequantities of HAPA are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HAPA may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced, and pIN vectors. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors(Pharmacia Biotech, Uppsala, Sweden) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-544.)

In cases where plant expression vectors are used, the expression ofsequences encoding HAPA may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al.(1991) Results Probl. Cell Differ. 17:85-105.) These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews. (See, e.g., Hobbs, S. or Murry,L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGrawHill, New York, N.Y.; pp. 191-196.)

An insect system may also be used to express HAPA. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HAPA may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of sequences encoding HAPA will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which HAPA may be expressed.(See, e.g., Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.91:3224-3227.)

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HAPA may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HAPA in infected host cells. (See, e.g., Logan, J.and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HAPA. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HAPA and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing HAPA can be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes and adenine phosphoribosyltransferase genes,which can be employed in tk⁻ or apr⁻ cells, respectively. (See, e.g.,Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980)Cell 22:817-823) Also, antimetabolite, antibiotic, or herbicideresistance can be used as the basis for selection. For example, dhfrconfers resistance to methotrexate; npt confers resistance to theaminoglycosides neomycin and G-418; and als or pat confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively. (See,e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570;Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry,supra.) Additional selectable genes have been described, e.g., trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine. (See,e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.85:8047-8051.) Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β glucuronidase and itssubstrate GUS, luciferase and its substrate luciferin. Green fluorescentproteins (GFP) (Clontech, Palo Alto, Calif.) are also used (See, e.g.,Chalfie, M. et al. (1994) Science 263:802-805.) These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHAPA is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HAPA can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HAPA under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HAPA and express HAPA may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding HAPA can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding HAPA. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HAPA to detect transformantscontaining DNA or RNA encoding HAPA.

A variety of protocols for detecting and measuring the expression ofHAPA, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HAPA is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HAPA includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HAPA,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech (Piscataway N.J.), Promega (Madison, Wis.), and U.S.Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules orlabels which may be used for ease of detection include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HAPA may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHAPA may be designed to contain signal sequences which direct secretionof HAPA through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding HAPA to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the HAPA encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing HAPA and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography. (IMIAC) (See, e.g., Porath, J. et al. (1992)Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage site provides ameans for purifying HAPA from the fusion protein. (See, e.g., Kroll, D.J. et al. (1993) DNA Cell Biol. 12:441453.)

Fragments of HAPA may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, T. E. (1984) Protein: Structures and MolecularProperties, pp. 55-60, W.H. Freeman and Co., New York, N.Y.) Proteinsynthesis may be performed by manual techniques or by automation.Automated synthesis may be achieved, for example, using the ABI 431APeptide synthesizer (PE Biosystems). Various fragments of HAPA may besynthesized separately and then combined to produce the full lengthmolecule.

Therapeutics

Chemical and structural homology exists between HAPA andbeta-alanine-pyruvate aminotransferase from rat (GI1944136). Inaddition, HAPA is expressed in tissues associated with immune disordersand cancer. Therefore, HAPA appears to play a role in immune disordersand cancer.

Therefore, in one embodiment, an antagonist of HAPA or a fragment orderivative thereof may be administered to a subject to treat or preventa cancer. Such cancers may include, but are not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma; and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds HMAP may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissues which express HMAP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HMAP may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

Therefore, in another embodiment, an antagonist of HMAP may beadministered to a subject to prevent or treat an immune disorder. Immunedisorders may include, but are not limited to AIDS, Addison's disease,adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis, andextracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma. In one aspect, anantibody which specifically binds HMAP may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissues which express HMAP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HMAP may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedescribed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HAPA may be produced using methods which are generallyknown in the art. In particular, purified HAPA may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HAPA. Antibodies to HAPA may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HAPA or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HAPA have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HAPA amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to HAPA may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HAPA-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HAPA mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HAPA and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HAPA epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingHAPA, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHAPA may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HAPA. Thus,complementary molecules or fragments may be used to modulate HAPAactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding HAPA.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencescomplementary to the polynucleotides of the gene encoding HAPA. (See,e.g., Sambrook, supra; and Ausubel, supra.) Genes encoding HAPA can beturned off by transforming a cell or tissue with expression vectorswhich express high levels of a polynucleotide, or fragment thereof,encoding HAPA. Such constructs may be used to introduce untranslatablesense or antisense sequences into a cell. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until they are disabled by endogenous nucleases. Transientexpression may last for a month or more with a non-replicating vector,and may last even longer if appropriate replication elements are part ofthe vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingHAPA. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHAPA.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HAPA. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HAPA,antibodies to HAPA, and mimetics, agonists, antagonists, or inhibitorsof HAPA. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HAPA, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HAPA or fragments thereof, antibodies of HAPA,and agonists, antagonists or inhibitors of HAPA, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD50/ED50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HAPA may beused for the diagnosis of disorders characterized by expression of HAPA,or in assays to monitor patients being treated with HAPA or agonists,antagonists, or inhibitors of HAPA. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for HAPA include methods which utilizethe antibody and a label to detect HAPA in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring HAPA, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HAPA expression. Normal or standard values for HAPAexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHAPA under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HAPA expressed in subjectsamples, control and disease, from biopsied tissues are compared withthe standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHAPA may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHAPA may be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of HAPA, and tomonitor regulation of HAPA levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HAPA or closely related molecules may be used to identifynucleic acid sequences which encode HAPA. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HAPA, alleles, orrelated sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe HAPA encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the HAPA gene.

Means for producing specific hybridization probes for DNAs encoding HAPAinclude the cloning of polynucleotide sequences encoding HAPA or HAPAderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HAPA may be used for the diagnosis ofa disorder associated with expression of HAPA. Examples of such adisorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma; and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immune disorders suchas AIDS, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, and extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections; and trauma. The polynucleotide sequences encoding HAPA maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered HAPA expression. Such qualitative orquantitative methods are well known in the art. The polynucleotidesequences encoding HAPA may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered HAPA expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HAPA may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHAPA may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding HAPA in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HAPA, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HAPA, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HAPA may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HAPA, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HAPA, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HAPAinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingHAPA may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HAPA on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., AT to 11q22-23, any sequences mapping to that area may representassociated or regulatory genes for further investigation. (See, e.g.,Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequenceof the subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc., amongnormal, carrier, or affected individuals.

In another embodiment of the invention, HAPA, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HAPAand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with HAPA, orfragments thereof, and washed. Bound HAPA is then detected by methodswell known in the art. Purified HAPA can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HAPA specificallycompete with a test compound for binding HAPA. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HAPA.

In additional embodiments, the nucleotide sequences which encode HAPAmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. LUNGTUT12 cDNA Library Construction

The LUNGTUT12 cDNA library was constructed from cancerous lung tissueobtained from a 70-year-old Caucasian female,(specimen #0319A) during alung lobectomy. Pathology for the left upper lobe lung tissue indicatedgrade 3 (of 4) adenocarcinoma, forming a mass which abuts but does notinvolve the pleura. Vascular invasion was present. The lobectomyspecimen contained no residual carcinoma. The bronchial margin was freeof involvement. Multiple (9) intrapulmonary peribronchial lymph nodeswere negative for tumor. Pathology for the left lower lobe lung tissueindicated lung parenchyma with focal subpleural fibrosis. Multiplesuperior mediastinal (2 left tracheo-bronchial), inferior mediastinal (8subcarinal; 4 inferior pulmonary ligament), 6 left lower lobe bronchus,and 4 left fissure lymph nodes were negative for tumor. Patient historyincluded tobacco abuse, depressive disorder, anxiety state, deviatednasal septum, malignant skin neoplasm, and a cesarean delivery. Previoussurgeries included an appendectomy. Family history included benignhypertension, cerebrovascular disease, and renal failure in the father;congestive heart failure, malignant colon neoplasm, and depressivedisorder in the mother; and primary malignant liver neoplasm in asibling.

The frozen tissues was homogenized and lysed in TRIZOL reagent (1 gmtissue/10 ml Trizol; Cat. #10296-028; GIBCO/BRL), a monoplastic solutionof phenol and guanidine isothiocyanate, using a POLYTRON homogenizer(PT-3000; Brinkmann Instruments, Westbury, N.Y.). After a briefincubation on ice, chloroform was added (1:5 v/v) and the lysate wascentrifuged. The upper chloroform layer was removed to a fresh tube andthe RNA extracted with isopropanol, resuspended in DEPC-treated water,and treated with DNase for 25 min at 37° C. RNA extraction andprecipitation were repeated as before. The mRNA was then isolated usingthe OLIGOTEX kit (QIAGEN, Chatsworth, Calif.) and used to construct thecDNA library.

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 plasmid kit (Catalog #26173, QIAGEN). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile TERRIFIC BROTH (Catalog #22711, GIBCO/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a MICROLAB 2200 (Hamilton) in combination with aDNA ENGINE thermal cyclers (PTC200 from MJ Research) and ABI PRISM 377DNA Sequencing systems (PE Biosystems).

III Homology Searching of cDNA Clones and Their Deduced Proteins

After the reading frame was determined, the nucleotide sequences of theSequence Listing as well as the amino acid sequences deduced from themwere used as query sequences against databases such as GenBank,SwissProt, BLOCKS, and Pima II. These databases, which containpreviously identified and annotated sequences, were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul (1993) supra, Altschul (1990) supra).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith et al. (1992, Protein Engineering 5:35-51),incorporated herein by reference, could have been used when dealing withprimary sequence patterns and secondary structure gap penalties. Thesequences disclosed in this application have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G, or T).

The BLAST approach, as detailed in Karlin et al. (supra) andincorporated herein by reference, searched for matches between a querysequence and a database sequence. BLAST evaluated the statisticalsignificance of any matches found, and reported only those matches thatsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10-25 for nucleotides and 10⁻¹⁴ forpeptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam);and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, F. M. et al. supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Genomics, Palo Alto, Calif.). This analysisis much faster than multiple membrane-based hybridizations. In addition,the sensitivity of the computer search can be modified to determinewhether any particular match is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact.Homologous molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HAPA occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of HAPA Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 3128715 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06software (National Biosciences, Plymouth, Minn.), or another appropriateprogram, to be about 22 to 30 nucleotides in length, to have a GCcontent of about 50% or more, and to anneal to the target sequence attemperatures of about 68° C. to about 72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationswas avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (PE Biosystems) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the DNA ENGINE thermal cycler(PTC200; M.J. Research), beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, with thefollowing parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (andholding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using a QIAQUICK kit (QIAGEN), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing 2×Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2×Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5′ regulatory sequences using the procedure above, oligonucleotidesdesigned for 5′ extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(NEN Life Sciences Products, Boston, Mass.). The labeledoligonucleotides are substantially purified using a SEPHADEX G-25superfine resin column (Amersham Pharmacia Biotech). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II (NEN Life Sciences Products).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots to film for severalhours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing or thermal, UV, mechanical, or chemical bonding procedures, or avacuum system. A typical array may be produced by hand or usingavailable methods and machines and contain any appropriate number ofelements. After hybridization, nonhybridized probes are removed and ascanner used to determine the levels and patterns of fluorescence. Thedegree of complementarity and the relative abundance of each probe whichhybridizes to an element on the microarray may be assessed throughanalysis of the scanned images.

In another alternative, full-length cDNAs or Expressed Sequence Tags(ESTs) comprise the elements of the microarray. Full-length cDNAs orESTs corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevent to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., U.V.cross-linking followed, by thermal and chemical and subsequent drying.(See, e.g., Schena, M. et al. (1995) Science 270:467-470; and Shalon, D.et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are preparedand used for hybridization to the elements on the substrate. Thesubstrate is analyzed by procedures described above.

Probe sequences for microarrays may be selected by screening a largenumber of clones from a variety of cDNA libraries in order to findsequences with conserved protein motifs common to genes coding forsignal sequence containing polypeptides. In one embodiment, sequencesidentified from cDNA libraries, are analyzed to identify those genesequences with conserved protein motifs using an appropriate analysisprogram, e.g., the Block 2 Bioanalysis Program (Incyte Genomics). Thismotif analysis program, based on sequence information contained in theSwiss-Prot Database and PROSITE, is a method of determining the functionof uncharacterized proteins translated from genomic or cDNA sequences.(See, e.g., Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221; andAttwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37:417-424.)PROSITE may be used to identify functional or structural domains thatcannot be detected using conserved motifs due to extreme sequencedivergence. The method is based on weight matrices. Motifs identified bythis method are then calibrated against the SWISS-PROT database in orderto obtain a measure of the chance distribution of the matches.

In another embodiment, Hidden Markov models (HMMs) may be used to findshared motifs, specifically consensus sequences. (See, e.g., Pearson, W.R. and D. J. Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; andSmith, T. F. and M. S. Waterman (1981) J. Mol. Biol. 147:195-197.) HMMswere initially developed to examine speech recognition patterns, but arenow being used in a biological context to analyze protein and nucleicacid sequences as well as to model protein structure. (See, e.g., Krogh,A. et al. (1994) J. Mol. Biol. 235:1501-1531; and Collin, M. et al.(1993) Protein Sci. 2:305-314.) HMMs have a formal probabilistic basisand use position-specific scores for amino acids or nucleotides. Thealgorithm continues to incorporate information from newly identifiedsequences to increase its motif analysis capabilities.

VIII. Complementary Polynucleotides

Sequences complementary to the HAPA-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HAPA. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of HAPA. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HAPA-encoding transcript.

IX. Expression of HAPA

Expression of HAPA is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., β-galactosidase upstreamof the cloning site, operably associated with the cDNA of interest.(See, e.g.,Sambrook, supra, pp. 404-433; and Rosenberg, M. et al. (1983)Methods Enzymol. 101:123-138.)

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of HAPA into bacterialgrowth media which can be used directly in the following assay foractivity.

X. Demonstration of HAPA Activity

The aminotransferase, HAPA, catalyze the transfer of an alpha-amino acidto an alpha-keto acid. The activity of the purified enzyme HAPA towardvarious amino and oxo acid substrates is determined under singleturnover conditions by monitoring the changes in the UV/VIS absorptionspectrum of the enzyme-bound cofactor. The reactions are conducted at 25° C. in 50 mM 4-methylmorpholine, pH 7.5, containing 9 uM (subunitconcentration) AspAT and the substrate. The half-reaction from aminoacid to oxo acid is followed by measuring the decrease in absorbance at360 nm and the increase in absorbance at 330 nm due to the conversion ofenzyme-bound pyridoxal 5′-phosphate to pyridoxamine 5′-phosphate; thereverse half-reaction is followed in an analogous manner. The PMP formof the enzymes is prepared by incubation of the PLP form with 1 mM PMPand 5 mM cysteine sulfinate for 30 minutes at 25 ° C. in the darkfollowed by SEPHADEX G-25 chromatography. The reactions are followedwith a Beckman 7400 DU spectrophotometer. With rapidly reactingsubstrates, a stopped-flow apparatus (a pbp-Spectra KineticMonochrometer 05-109 from Applied Photophysics) with a cuvette of 1-cmpath length and a dead time of 2 ms is used. Steady-state aspartateaminotransferase activity is measured in a coupled assay with malatedehydrogenase in 50 mM 4-methylmorpholine, pH 7.5, at 25 ° C. in thepresence of 20 mM 2-oxoglutarate plus 20 mM or 150 mM L-aspartate forwild-type enzyme or mutant AspATs, respectively.

XI. Production of HAPA Specific Antibodies

HAPA substantially purified using PAGE electrophoresis (see, e.g.,Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or otherpurification techniques, is used to immunize rabbits and to produceantibodies using standard protocols. The HAPA amino acid sequence isanalyzed using LASERGENE software (DNASTAR Inc) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Methods for selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions are well described in the art.(See, e.g., Ausubel et al. supra, ch. 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) byreaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) toincrease immunogenicity. (See, e.g., Ausubel et al. supra.) Rabbits areimmunized with the oligopeptide-KLH complex in complete Freund'sadjuvant. Resulting antisera are tested for antipeptide activity, forexample, by binding the peptide to plastic, blocking with 1% BSA,reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XII. Purification of Naturally Occurring HAPA Using Specific Antibodies

Naturally occurring or recombinant HAPA is substantially purified byimmunoaffinity chromatography using antibodies specific for HAPA. Animmunoaffinity column is constructed by covalently coupling anti-HAPAantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing HAPA are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HAPA (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HAPA binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HAPAis collected.

XIII. Identification of Molecules Which Interact with HAPA

HAPA, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HAPA, washed, and anywells with labeled HAPA complex are assayed. Data obtained usingdifferent concentrations of HAPA are used to calculate values for thenumber, affinity, and association of HAPA with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

3 450 amino acids amino acid single linear LUNGTUT12 3128715 1 Met AlaAla Asp Gln Arg Pro Lys Ala Asp Thr Leu Ala Leu Arg Gln 1 5 10 15 ArgLeu Ile Ser Ser Ser Cys Arg Leu Phe Phe Pro Glu Asp Pro Val 20 25 30 LysIle Val Arg Ala Gln Gly Gln Tyr Met Tyr Asp Glu Gln Gly Ala 35 40 45 GluTyr Ile Asp Cys Ile Ser Asn Val Ala His Val Gly His Cys His 50 55 60 ProLeu Val Val Gln Ala Ala His Glu Gln Asn Gln Val Leu Asn Thr 65 70 75 80Asn Ser Arg Tyr Leu His Asp Asn Ile Val Asp Tyr Ala Gln Arg Leu 85 90 95Ser Glu Thr Leu Pro Glu Gln Leu Cys Val Phe Tyr Phe Leu Asn Ser 100 105110 Gly Ser Glu Ala Asn Asp Leu Ala Leu Arg Leu Ala Arg His Tyr Thr 115120 125 Gly His Gln Asp Val Val Val Leu Asp His Ala Tyr His Gly His Leu130 135 140 Ser Ser Leu Ile Asp Ile Ser Pro Tyr Lys Phe Arg Asn Leu AspGly 145 150 155 160 Gln Lys Glu Trp Val His Val Ala Pro Leu Pro Asp ThrTyr Arg Gly 165 170 175 Pro Tyr Arg Glu Asp His Pro Asn Pro Ala Met AlaTyr Ala Asn Glu 180 185 190 Val Lys Arg Val Val Ser Ser Ala Gln Glu LysGly Arg Lys Ile Ala 195 200 205 Ala Phe Phe Ala Glu Ser Leu Pro Ser ValGly Gly Gln Ile Ile Pro 210 215 220 Pro Ala Gly Tyr Phe Ser Gln Val AlaGlu His Ile Arg Lys Ala Gly 225 230 235 240 Gly Val Phe Val Ala Asp GluIle Gln Val Gly Phe Gly Arg Val Gly 245 250 255 Lys His Phe Trp Ala PheGln Leu Gln Gly Lys Asp Phe Val Pro Asp 260 265 270 Ile Val Thr Met GlyLys Ser Ile Gly Asn Gly His Pro Val Ala Cys 275 280 285 Val Ala Ala ThrGln Pro Val Ala Arg Ala Phe Glu Ala Thr Gly Val 290 295 300 Glu Tyr PheAsn Thr Phe Gly Gly Ser Pro Val Ser Cys Ala Val Gly 305 310 315 320 LeuAla Val Leu Asn Val Leu Glu Lys Glu Gln Leu Gln Asp His Ala 325 330 335Thr Ser Val Gly Ser Phe Leu Met Gln Leu Leu Gly Gln Gln Lys Ile 340 345350 Lys His Pro Ile Val Gly Asp Val Arg Gly Val Gly Leu Phe Ile Gly 355360 365 Val Asp Leu Ile Lys Asp Glu Ala Thr Arg Thr Pro Ala Thr Glu Glu370 375 380 Ala Ala Tyr Leu Val Ser Arg Leu Lys Glu Asn Tyr Val Leu LeuSer 385 390 395 400 Thr Asp Gly Pro Gly Arg Asn Ile Leu Lys Phe Lys ProPro Met Cys 405 410 415 Phe Ser Leu Asp Asn Ala Arg Gln Val Val Ala LysLeu Asp Ala Ile 420 425 430 Leu Thr Asp Met Glu Glu Lys Val Arg Ser CysGlu Thr Leu Arg Leu 435 440 445 Gln Pro 450 1786 base pairs nucleic acidsingle linear LUNGTUT12 3128715 2 CGCCCGCGCG GCGACGTCTC CGCGAGGCGTCACGGCACCG ACTGACGGCC ACCCACCAT 60 GCCGCAGACC AGCGCCCGAA GGCCGACACCCTGGCCCTGA GGCAACGGCT CATCAGCT 120 TCCTGCAGAC TCTTTTTTCC CGAGGATCCTGTTAAGATTG TCCGGGCCCA AGGGCAGT 180 ATGTACGATG AACAGGGGGC AGAATACATCGATTGCATCA GCAATGTGGC GCACGTTG 240 CACTGCCACC CTCTCGTGGT CCAAGCAGCACATGAGCAGA ACCAGGTGCT CAACACCA 300 AGCCGGTACC TGCATGACAA CATCGTGGACTATGCGCAGA GGCTGTCAGA GACCCTGC 360 GAGCAGCTCT GTGTGTTCTA TTTCCTGAATTCTGGGTCAG AAGCCAATGA CCTGGCCC 420 AGGCTGGCTC GCCACTACAC GGGACACCAGGACGTGGTGG TATTAGATCA TGCGTATC 480 GGCCACCTGA GCTCCCTGAT TGACATCAGTCCCTACAAGT TCCGCAACCT GGATGGCC 540 AAGGAGTGGG TCCACGTGGC ACCTCTCCCAGACACCTACC GGGGCCCCTA CCGGGAGG 600 CACCCCAACC CAGCTATGGC CTATGCCAACGAGGTGAAAC GTGTGGTCAG CAGTGCAC 660 GAGAAGGGCA GGAAGATTGC AGCCTTCTTCGCTGAGTCTC TGCCCAGTGT GGGAGGGC 720 ATCATTCCCC CTGCTGGCTA CTTCTCCCAAGTGGCAGAGC ACATCCGCAA GGCCGGAG 780 GTCTTTGTTG CAGATGAGAT CCAGGTTGGCTTTGGCCGGG TAGGCAAGCA CTTCTGGG 840 TTCCAGCTCC AGGGAAAAGA CTTCGTCCCTGACATCGTCA CCATGGGCAA GTCCATTG 900 AACGGCCACC CTGTTGCCTG CGTGGCCGCAACCCAGCCTG TGGCGAGGGC ATTTGAAG 960 ACCGGCGTTG AGTACTTCAA CACGTTTGGGGGCAGCCCAG TGTCCTGCGC TGTGGGG 1020 GCCGTCCTGA ATGTCTTGGA GAAGGAGCAGCTCCAGGATC ATGCCACCAG TGTAGGC 1080 TTCCTGATGC AGCTCCTCGG GCAGCAAAAAATCAAACATC CCATCGTCGG GGATGTC 1140 GGTGTTGGGC TCTTCATTGG TGTGGATCTGATCAAAGATG AGGCCACAAG GACACCA 1200 ACTGAAGAGG CTGCCTACTT GGTATCAAGGCTGAAGGAGA ACTACGTTTT GCTGAGC 1260 GATGGCCCTG GGAGGAACAT CCTGAAGTTTAAGCCCCCAA TGTGCTTCAG CCTGGAC 1320 GCACGGCAGG TGGTGGCAAA GCTGGATGCCATTCTGACTG ACATGGAAGA GAAGGTG 1380 AGTTGTGAAA CGCTGAGGCT CCAGCCCTAAGCCAGCCCTG CTCTGCCTAA GTGTACT 1440 GAAGAAACTC ATCTCATCCA AATACACGCTATTGAGAAGG CGAGCCTGAC CTCCCTC 1500 CAGATAAAGT CAGCTTTCAG AGGCTCAGGGTGGGGGGGCC TGCCCGAGGC CATAATG 1560 CCCACCCCCT CCTCCTAACC ACTGGTCTGTTGGAATAACC CAGATGTCTG CATCCCC 1620 AGTCAGTCAA TTTCCTTTCT GTCCACTGGGGGTGGAATGG GGTAGGGTGG GATACTT 1680 AGTGCTCCTG CTTAAATAAA TTAGACCAGACCAGTGTATT TCTAAAGAAA ATCCTGA 1740 GCACACCCAT TAAAAATAGT ACATTTTACAGTGAAAAAAA AAAAAA 1786 512 amino acids amino acid single linear GenBank1944136 3 Met Ser Leu Ala Trp Arg Thr Leu Gln Lys Ala Phe Tyr Leu GluThr 1 5 10 15 Ser Leu Arg Ile Leu Gln Met Arg Pro Ser Leu Ser Cys AlaSer Arg 20 25 30 Ile Tyr Val Pro Lys Leu Thr Leu His Thr Lys His Asn MetPro Pro 35 40 45 Cys Asp Phe Ser Pro Glu Lys Tyr Gln Ser Leu Ala Tyr AsnHis Val 50 55 60 Leu Glu Ile His Lys Gln His Leu Ser Pro Val Asn Thr AlaTyr Phe 65 70 75 80 Gln Lys Pro Leu Leu Leu His Gln Gly His Met Glu TrpLeu Phe Asp 85 90 95 Ser Glu Gly Asn Arg Tyr Leu Asp Phe Phe Ser Gly IleVal Thr Val 100 105 110 Gly Val Gly His Cys His Pro Lys Val Thr Ala ValAla Lys Lys Gln 115 120 125 Met Asp Arg Leu Trp His Thr Ser Ser Val PhePhe His Ser Pro Met 130 135 140 His Glu Tyr Ala Glu Arg Leu Ser Ala LeuLeu Pro Glu Pro Leu Lys 145 150 155 160 Val Ile Phe Leu Val Asn Ser GlySer Glu Ala Asn Asp Leu Ala Met 165 170 175 Val Met Ala Arg Ala Tyr SerAsn His Thr Asp Ile Ile Ser Phe Arg 180 185 190 Gly Ala Tyr His Gly CysSer Pro Tyr Thr Leu Gly Leu Thr Asn Val 195 200 205 Gly Ile Tyr Lys MetLys Val Pro Ser Thr Ile Ala Cys Gln Ser Thr 210 215 220 Met Cys Pro AspVal Phe Arg Gly Pro Trp Gly Gly Ser His Cys Arg 225 230 235 240 Asp SerPro Val Gln Thr Val Arg Lys Cys Ser Cys Ala Pro Asp Gly 245 250 255 CysGln Ala Lys Glu Arg Tyr Ile Glu Gln Phe Lys Asp Thr Leu Asn 260 265 270Thr Ser Val Ala Thr Ser Ile Ala Gly Phe Phe Ala Glu Pro Ile Gln 275 280285 Gly Val Asn Gly Val Val Gln Tyr Pro Lys Glu Phe Leu Lys Glu Ala 290295 300 Phe Ala Leu Val Arg Glu Arg Gly Gly Val Cys Ile Ala Asp Glu Val305 310 315 320 Gln Thr Gly Phe Gly Arg Leu Gly Ser His Phe Trp Gly PheGln Thr 325 330 335 His Asp Thr Met Pro Asp Ile Val Thr Met Ala Lys GlyIle Gly Asn 340 345 350 Gly Phe Pro Met Ala Ala Val Val Thr Thr Pro GluIle Ala Ser Ser 355 360 365 Leu Ala Lys His Leu His His Phe Ser Thr PheGly Gly Ser Pro Leu 370 375 380 Ala Cys Ala Ile Gly Ser Ala Val Leu GluVal Ile Glu Glu Glu Asn 385 390 395 400 Leu Gln Arg Asn Ser Gln Glu ValGly Thr Tyr Met Leu Leu Lys Phe 405 410 415 Ala Lys Leu Arg Asp Glu PheAsp Ile Val Gly Asp Val Arg Gly Lys 420 425 430 Gly Leu Met Val Gly IleGlu Met Val Gln Asp Lys Ile Ser Arg Gln 435 440 445 Pro Leu Pro Lys ThrGlu Val Asn Gln Ile His Glu Asp Cys Lys Asp 450 455 460 Met Gly Leu LeuVal Gly Arg Gly Gly Asn Phe Ser Gln Thr Phe Arg 465 470 475 480 Ile AlaPro Pro Met Arg Val Thr Lys Leu Glu Val Asp Phe Ala Phe 485 490 495 GluVal Phe Arg Ser Ala Leu Thr Gln His Met Glu Arg Arg Ala Lys 500 505 510

What is claimed is:
 1. A purified polypeptide comprising an amino acidsequence selected from the group consisting of: a) an amino acidsequence of SEQ ID NO:1, b) an amino acid sequence having at least 90%sequence identity to the sequence of SEQ ID NO:1, and which retainsbeta-alanine-pyruvate aminotransferase activity, and c) abiologically-active fragment of the amino acid sequence of SEQ ID NO:1.2. An isolated polypeptide of claim 1, having a sequence of SEQ ID NO:1.3. A composition comprising an effective amount of a polypeptide ofclaim 1 and a pharmaceutically acceptable excipient.
 4. A composition ofclaim 3, wherein the polypeptide has the sequence of SEQ ID NO:1.
 5. Amethod for screening a compound for effectiveness as an agonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingagonist activity in the sample.
 6. A method for screening a compound foreffectiveness as an antagonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting antagonist activity in the sample.
 7. Amethod for using a polypeptide for screening a plurality of molecules orcompounds for a molecule or compound which specifically binds thepolypeptide, the method comprising: a) combining the polypeptide ofclaim 1 with a plurality of molecules or compounds under conditions toallow specific binding, and b) detecting specific binding between thepolypeptide and the molecule or compound, thereby identifying a moleculeor compound that specifically binds the polypeptide.
 8. A method ofusing a polypeptide to purify a molecule or compound which specificallybinds the polypeptide from a sample, the method comprising: a) combiningthe polypeptide of claim 1 with a sample under conditions to allowspecific binding; b) recovering the bound polypeptide; and c) separatingthe polypeptide from the molecule or compound, thereby obtainingpurified molecule or compound.
 9. An isolated immunogenic fragment ofthe amino acid sequence of SEQ ID NO:1.